Crop Science 43:1072-1075 (2003)
© 2003 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Field Efficacy Assessment of Transgenic Roundup Ready Wheat
H. Zhou*,
J. D. Berg,
S. E. Blank,
C. A. Chay,
G. Chen,
S. R. Eskelsen,
J. E. Fry,
S. Hoi,
T. Hu,
P. J. Isakson,
M. B. Lawton,
S. G. Metz,
C. B. Rempel,
D. K. Ryerson,
A. P. Sansone,
A. L. Shook,
R. J. Starke,
J. M. Tichota and
S. A. Valenti
Monsanto Company, Mail Zone: E2SI, 800 N. Lindbergh Blvd, St. Louis, MO 63167
* Corresponding author (hua-ping.zhou{at}monsanto.com)
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ABSTRACT
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Herbicide tolerant crops provide farmers access to a new weed control option of nonselective herbicide such as Roundup1. A wheat transgenic event 33391 was produced via Agrobacterium-mediated transformation of a donor cultivar Bobwhite wheat (Triticum aestivum L.) and was identified as a commercial candidate to develop Roundup Ready wheat2. The objective of this study was to assess field efficacy of the transgenic event in spring wheat production regions in North America. Transgenic event 33391 was tested in field trials at 14 locations in 1999, 13 locations in 2000, and 14 locations in 2001. All trials were split-plot designs with multiple rates of Roundup treatment. No vegetative or reproductive damage was observed with the application of 4 L ha-1 Roundup at the 3- to 5-leaf stages. No yield reduction was observed with Roundup treatment. The transgenic event with or without Roundup application yielded as high as the nontransgenic Bobwhite. These results indicate that the wheat transgenic event 33391 has at least 2x tolerance to the nonselective herbicide Roundup.
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INTRODUCTION
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WHEAT is the most widely grown cereal crop in the world. Genetic engineering is of significant interest to improving productivity and grower profitability. Because of inefficient transformation and regeneration of viable plants and low expression of transgenes, genetic engineering of wheat has been difficult (Vasil et al., 1992; Vasil et al., 2001). Recent progress in transformation methods has made it possible to transfer and express genes from other species into wheat to achieve agronomically desirable traits, such as herbicide resistance (Cheng et al., 1997; Zhou et al., 1995). There are approximately 20 million hectares of spring wheat planted in North America each year. Over 80% of the planted hectares are treated with at least one herbicide during the growing season to control weeds (USDA/NASS, 2001). Most of the current herbicides have a narrow spectrum of weed control. Some of them persist in soil, which may limit crop rotation on the same field the following season. Extensive use of common herbicides, such as the ACCase and ALS inhibitors, has resulted in the selection for weed resistance to these herbicides (Heap, 2001). Roundup, which contains the active ingredient glyphosate, has a unique mode of action that is different from other products on the market. As a nonselective herbicide, Roundup provides effective control of a broad spectrum of weed species by blocking the production of aromatic amino acids (phenylalanine, tyrosine, and tryptophan), which are essential to plant growth and development.
Aromatic amino acids are synthesized through the shikimate pathway, where EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) is a critical enzyme. Roundup inhibits the endogenous plant EPSPS. An EPSPS gene (aroA:CP4) resistant to Roundup was isolated from a soil-borne Agrobacterium sp. strain CP4 (Barry et al., 1992). When targeted to chloroplasts by a chloroplast transit peptide the aroA:CP4 EPSPS provides tolerance to the nonselective herbicide Roundup in soybean (Glycine max (L.) Merr.) (Padgette et al., 1995) and cotton (Gossypium spp.) (Nida et al., 1996).
Early transgenic events containing the aroA:CP4 gene driven by the CaMV 35S promoter had excellent vegetative tolerance but were partially sterile (data not published). This could be due to limited expression of the CaMV 35S promoter in reproductive tissues. To achieve adequate expression of the aroA:CP4 gene, a two promoter strategy was deployed to develop glyphosate tolerant wheat. One aroA:CP4 gene is under the control of the enhanced CaMV 35S promoter (Kay et al., 1987) and a second aroA:CP4 gene is under the control of the rice actin 1 promoter (McElroy et al., 1990), which has been shown to drive expression of a reporter gene in reproductive tissues (Zhang et al., 1991). We expected that these two promoters would complement each other to provide constitutive expression in all plant organs and throughout all growth stages.
The product concept of Roundup Ready wheat is to control annual weeds and suppress perennial weeds in wheat production fields. This is to be achieved by using 2 L ha-1 Roundup at 3- to 5-leaf stages for spring wheat and 2 L ha-1 Roundup during the fall followed by 2 L ha-1 in the spring for winter wheat. Our initial goal was to achieve the Roundup tolerance in spring wheat with a 2x safety factor. Over a thousand of wheat transgenic events with the aroA:CP4 genes were produced at Monsanto. After years of tolerance test, genetic analysis, and molecular characterization, several events were identified as commercial candidates to develop Roundup Ready wheat. The objective of this study was to assess field efficacy of the lead event 33391 in spring wheat production regions in the USA and Canada.
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MATERIALS AND METHODS
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Production of Roundup Ready Transgenic Wheat Plants
Over 50 expression vectors were constructed to optimize expression of the aroA:CP4 gene in wheat plants. Each vector had different genetic elements (promoters, introns, and 3' UTR) or combinations of those elements. One of the most successful plant expression vectors was binary vector PV-TXGT10, which harbored two separate aroA:CP4 expression cassettes within the same T-DNA (Fig. 1). One cassette had the aroA:CP4 gene driven by the rice actin 1 promoter and the other by the CaMV enhanced 35S promoter. Both aroA:CP4 genes were targeted to the chloroplast by the Arabidopsis thaliana EPSPS chloroplast transit peptide translationally fused to the N terminus of the CP4 EPSPS protein (Shah et al., 1986). Additional regulatory elements included the first intron of the rice acrin 1 promoter (McElroy et al., 1990), the 3' non-translated region of the nopoline synthase gene from Agrobacterium tumefaciens (Fraley et al., 1983), and an intron from the maize hsp70 gene (Brown and Santino, 1994).
The T-DNA was introduced into actively growing wheat tissues derived from immature embryos through an Agrobacterium-mediated transformation protocol (Cheng et al., 1997). A hard red spring wheat cultivar Bobwhite was used as the recipient genotype since it has high transformation and regeneration efficiencies using the current transformation protocol. Although many more events were generated from the other vectors, most of those had low gene expression and were eliminated from further evaluation. From the binary vector PV-TXGT10, 131 independent transgenic events were produced. These events were tested and characterized for several generations in growth chambers and in laboratories to ensure single insert, single copy of the T-DNA, stable expression across generations, transgene placement on a desirable chromosome, intactness of the insert, and absence of extra T-DNA fragment and vector backbone. Transgenic event 33391 meets these criteria and contains the sequence between the right and left borders of T-DNA from the PV-TXGT10 binary vector.
Field Efficacy Assessment
Selected transgenic events were tested under field conditions to determine efficacy of Roundup tolerance in typical spring wheat production regions in the USA and Canada during the summers of 1999, 2000, and 2001. In 1999, there were eight sites in the USA and eight sites in Canada, but two U.S. sites were lost because of inclement weather conditions. In 2000, there were eight sites in the USA and six sites in Canada, but one site was lost in Canada. In 2001, there were eight sites in the USA and six sites in Canada. All sites are listed by year and by country in Table 1.
In 1999, all trials in the USA and Canada had two Roundup treatments and one unsprayed control. The treatment rates were 0, 2 L ha-1, and 4 L ha-1 Roundup or approximately 0, 0.9 kg ha-1, and 1.8 kg ha-1 glyphosate acid (Roundup Ultra was used in the USA and Roundup Transorb in Canada). In 2000, an additional high rate treatment of 8 L ha-1 or approximately 3.6 kg ha-1 glyphosate acid was added at all sites. In 2001, all trials in the USA received 0, 4 L ha-1, and 8 L ha-1 Roundup, whereas trials in Canada received 0, 2 L ha-1 and 8 L ha-1 Roundup. The application of Roundup was targeted at the 3- to 5-leaf stages with one or two tillers per plant. The trials were kept weed free with a locally recommended conventional grass herbicide as base treatment. The base treatment was applied 7 to 10 d before Roundup application. Carrying volume for all herbicide treatments was approximately 100 L ha-1.
In 1999, event 33391 and four other Roundup Ready wheat commercial candidates were tested in all trials. At the end of season, three events were dropped on the basis of data from the field trials and molecular analysis. In 2000, event 33391 and one other commercial candidate were tested in all trials. In 2001, event 33391 and five new commercial candidates were tested at all sites. The wild-type nontransgenic Bobwhite was used as a check in all trials except those in the USA in 2000 to determine equivalence of transgenic and nontransgenic wheat and to confirm the effectiveness of Roundup treatments. Transgenic seeds of event 33391 were from an R4 generation produced in Hawaii during the winter of 1998. Seeds of the nontransgenic Bobwhite were also produced in Hawaii during the same season to ensure uniform seed quality.
All trials were split-plot designs with Roundup treatments or genotypes as the main plots. Since the other transgenic events included in the trials were not presented in this paper and the nontransgenic Bobwhite died after Roundup treatment, the split-plot designs collapsed into randomized complete block (RCB) designs. Therefore, the data were analyzed and presented as two separate RCB designs, one focusing on Roundup treatment effect on the transgenic event and the other on genotype effect between the transgenic event 33391 and the nontransgenic Bobwhite. Plot sizes were 6 m long and 1.5 m wide, and were trimmed to 4.5 m long before harvest. All U.S. trials had six replications in 1999 and 2000 and four replications in 2001. Canadian trials had four replications in 1999 and 2000 and three replications in 2001. Seeding rate was approximately 80 kg ha-1 or 70 g per plot. Interrow space varied between sites depending on cone planters available at each site.
Vegetative tolerance was evaluated and recorded 7 and 14 d after the Roundup applications, whereas reproductive tolerance was observed 14 d after anthesis. Disease response, phenotype, growth and development stages, and other agronomic traits were evaluated and recorded during the growing season. All trials were harvested and grain yield was recorded. The data were analyzed to determine event performance and Roundup treatment effects. Although other transgenic events beside the 33391 were included in the tolerance trials throughout the three years, those events were discontinued on the basis of field performance and molecular analysis. Only data from the transgenic event 33391 are reported in this paper.
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RESULTS AND DISCUSSION
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Conventional wheat cultivars are susceptible to Roundup at rates as low as 0.2 L ha-1 under field conditions (Zhou and Reeder, 1998, unpublished). In the tolerance trials from 1999 through 2001, the nontransgenic Bobwhite died within 7 d after Roundup application, whereas no chlorosis, necrosis, or growth reduction was observed in Roundup treated transgenic event 33391. Reproductive damage, often observed as bleached spikes or gapping (florets kept open after anthesis because of sterility), was not observed at any location with any Roundup treatment on the transgenic event. No differences in disease response, maturity, or robustness of plants were observed among the treatments or between the transgenic event and the wild-type nontransgenic Bobwhite throughout the growing season (data not shown).
Grain yield is another measurement of treatment effects by Roundup since even a minor vegetative or reproductive damage from the treatments could cause yield reduction. Grain yield was analyzed across all locations within each year and the analysis of variance is presented in Table 2. Significant yield differences were observed among sites for all three years. This was expected because of the wide environmental differences across the spring wheat production regions. Yield in the Pacific Northwest sites was twice as high as those in the Great Plains and most sites in Canada. Treatment effects were also observed among Roundup rates in some years. In 1999, the 2 and 4 L ha-1 Roundup treatments yielded 2.74 and 2.70 Mg ha-1, respectively. The non-Roundup treatment (0 L ha-1) only yielded 2.56 Mg ha-1, which is statistically lower than the Roundup treatments at the 5% probability level (Table 3). It is not clear why Roundup application led to higher yield, which warrants additional studies to confirm and to explain the difference. In 2000, the Roundup treatments yielded 4.03 to 4.08 Mg ha-1, which is slightly higher but not statistically different from the 3.86 Mg ha-1 of the non-Roundup treatment (Table 3). In 2001, significant difference among Roundup treatments was observed in Canada. The 0 and 2 L ha-1 Roundup treatments had similar grain yield (2.58 and 2.66 Mg ha-1), but the 8 L ha-1 treatment significantly out-yielded (2.90 Mg ha-1) the other two treatments. In the USA, yield of the 2001 trials varied from 3.49 to 3.51 Mg ha-1 among different treatments, which is not statistically significant.
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Table 2. Analysis of variance of grain yields from Roundup Ready wheat field efficacy trials in 1999, 2000, and 2001.
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Table 3. Means and standard errors of grain yield (Mg ha-1) from Roundup Ready wheat field efficacy trials in 1999, 2000, and 2001.
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Treatment x site interactions were significant at the 5% level in 2000, where the majority of the interactions were explained by the higher yields of the 4 and 8 L ha-1 Roundup treatments at the Lethbridge and Portage sites. The same treatments had lower yield at the Cassie site. In 2001, all trials in the USA had Roundup treatments as blocks. This design is effective to avoid Roundup drift but failed to partition the variance of treatment by site interactions from the error. Data from the 2001 U.S. trials were analyzed and presented separately in Tables 2 and 3. Treatment x site interactions were not significant in 1999 and 2001 in Canada (Table 2).
The transgenic Roundup Ready wheat must show equivalent performance as nontransgenic wheat under environment conditions where Roundup treatment is not required because of low weed pressure or cannot be applied because of operational difficulties. To determine such equivalence, the wild-type nontransgenic Bobwhite was included as a check in most trials except those in the USA in 2000. Because of Roundup damage from spray drift, nontransgenic Bobwhite at the Coalhurst site in 1999 and the Lethbridge site in 2000 was excluded from the analysis. Direct comparisons between the transgenic event and nontransgenic Bobwhite were made at 13 sites in 1999, four sites in 2000, and 14 sites in 2001. At the 0 L ha-1 treatment, no morphological or physiological difference was observed between the transgenic event 33391 and nontransgenic Bobwhite at any site in any year. Yields of the non-Roundup treatment plots were analyzed across sites within each year and the analysis is presented in Table 4. The yield difference between the genotypes was insignificant in 1999. In 2000, the transgenic event out-performed the nontransgenic Bobwhite by a significant margin (Table 5). Among three of the four sites, the transgenic event had higher yield than the nontransgenic Bobwhite, whereas the 4th site had virtually identical yield between them. In 2001, the genotypic difference was not significant, but genotype x site interactions were significant at the 1% level. These interactions were due to higher yield of the transgenic event at the Lacombe and Portage sites but lower yield at the Lethbridge and Minto sites. Genotype x site interactions were not significant at the 5% level in 1999 and 2000.
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Table 5. Yield (Mg ha-1) comparison of transgenic event 33391 and the wild-type nontransgenic Bobwhite when Roundup was not applied.
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Results from the 3-yr field efficacy studies demonstrated that the transgenic event 33391 tolerated at least 4 L ha-1 Roundup, which is twice the commercial target rate. The study also indicated that neither the transformation process nor the aroA:CP4 gene placement resulted in detrimental effects to agronomic performance of the Roundup Ready transgenic event. The efficacy of Roundup tolerance and the equivalence between the transgenic event and the wild-type nontransgenic Bobwhite meet the commercial criteria established earlier. Development of Roundup Ready wheat will provide farmers access to a new weed control option that is cost-effective, flexible, and reliable at a wide range of environment conditions.
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ACKNOWLEDGMENTS
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The authors are grateful to M. Radionenko, F. Lu, and X. Feng for the production of Roundup resistant transgenic wheat plants. Thanks also to T. Armstrong, M. McCann, and K. Boddy for their support with field trials and to P. Hunter for handling the application of field release notifications and USDA-APHIS compliance. Many others have made considerable contributions to this project and are greatly appreciated.
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NOTES
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Roundup and Roundup Ready are trademarks of Monsanto Company.
Received for publication March 21, 2002.
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ABSTRACT
INTRODUCTION
